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The Core and Accessory Genomes of Burkholderia pseudomallei: Implications for Human Melioidosis

Natural isolates of Burkholderia pseudomallei (Bp), the causative agent of melioidosis, can exhibit significant ecological flexibility that is likely reflective of a dynamic genome. Using whole-genome Bp microarrays, we examined patterns of gene presence and absence across 94 South East Asian strains isolated from a variety of clinical, environmental, or animal sources. 86% of the Bp K96243 reference genome was common to all the strains representing the Bp “core genome”, comprising genes largely involved in essential functions (eg amino acid metabolism, protein translation). In contrast, 14% of the K96243 genome was variably present across the isolates. This Bp accessory genome encompassed multiple genomic islands (GIs), paralogous genes, and insertions/deletions, including three distinct lipopolysaccharide (LPS)-related gene clusters. Strikingly, strains recovered from cases of human melioidosis clustered on a tree based on accessory gene content, and were significantly more likely to harbor certain GIs compared to animal and environmental isolates. Consistent with the inference that the GIs may contribute to pathogenesis, experimental mutation of BPSS2053, a GI gene, reduced microbial adherence to human epithelial cells. Our results suggest that the Bp accessory genome is likely to play an important role in microbial adaptation and virulence.

Introduction
Melioidosis is a potentially fatal infectious disease of humans and animals caused by the Gram-negative bacterium Burkholderia pseudomallei (Bp) [1]. An environmental saphrophyte found in South East Asia, Bp infections in endemic areas may be responsible for up to 20% of deaths due to septicemia [2,3], and Bp has been designated a Category B biothreat agent [4]. A wide spectrum of disease symptoms are associated with melioidosis often leading to late diagnosis and treatment [5]. Commonly presenting as an acute septicemic illness, chronic Bp infection is also well recognized which can be confused with TB or malignancy [6]. Besides humans, Bp has a broad host range and can infect nematodes, amoebae, dolphins, birds, swine, sheep, and gorillas [7][8][9][10][11]. Bp can also be isolated from diverse environmental sources such as soil, water, and air [12][13][14][15][16][17]. Identifying the molecular factors responsible for this tremendous ecologic flexibility may improve our understanding of microbial survival and adaptation, and suggest novel diagnostic and treatment strategies for melioidosis.
The phenotypic versatility of Bp is likely to be underpinned by the presence of a highly dynamic genome. For example, lateral gene transfer events may cause large-scale variations in genome content [18]. The portion of the genome that is variably present between individual strains is often termed the ''accessory genome'', to distinguish these genes from genes common to all strains in a population and involved in essential functions (the ''core'' genome). In several microbial species, accessory genes have been shown to play key roles in host adaptation and, in the case of Bp, the accessory genome may contribute to virulence and antibiotic resistance [19]. Interestingly, previous studies indicate that in Bp, gene loss, as well as gene acquisition events, can both cause phenotypic shifts towards virulence. For example, comparisons between Bp and B. thailandensis, an avirulent closely related species, have shown that an important evolutionary step in the development of Bp pathogenicity was the loss of an anti-virulence arabinose assimilation cluster [20,21]. Such findings thus raise a compelling need to accurately define the core and accessory genomes of Bp.
In other c proteobacteria genera (E. coli, Pseudomonas, Vibrio), the accessory genome can encompass up to 20% of all genomic content, and similar percentages may also hold for Burkholderia spp. [22][23][24][25]. However, to date, comprehensive qualitative and quantitative studies of the core and accessory genome in Bp have not been carried out, and the full extent to which gene content differences contribute to virulence in Bp is still unclear. While some previous studies have attempted to explore these issues, they have not incorporated data from the whole genome [19,[26][27][28], or have used only a very small sample of strains [29,30]. In this study, we performed a detailed array-based comparative genomic hybridization (aCGH) analysis of close to 100 clinical, animal and environmental Bp isolates from South East Asia. To our knowledge, this is the first time a whole genome comparative study has been applied to such a large Bp strain cohort. We found that 86% of the reference Bp K96243 genome was present in all the strains, while the remaining 14% was variably present across the strain panel. Surprisingly, isolates associated with human melioidosis exhibited a tendency to harbor certain GIs compared to isolates from either animal or environmental sources, suggesting that genes on these mobile elements might facilitate colonization of the human host. Taken collectively, our results support the notion that the Bp accessory genome may play a central role in adaptation and virulence. Besides providing important evidence concerning genes likely involved in Burkholderia pathogenesis, this study also raises the possibility of targeting molecular diagnostics to specific Bp accessory regions for monitoring the presence of human-virulent variants in the environment.

Genome-wide Identification of Core and Accessory Genes in Bp Isolates
Using a previously validated Bp K96243 DNA microarray [30,31], we generated aCGH profiles for ninety-four Bp strains isolated from human patients, animals, and environmental soils in Singapore, Malaysia or Thailand (Table S1). We applied a Gaussian Mixture Model (GMM) to the aCGH data and identified 750 out of 5369 genes (14%) as being variably present across the strain panel (see Methods and Figure S1). The variability of the 750 genes was experimentally validated by several independent methods, including bioinformatic comparisons to previouslyknown variable genes, comparisons against publicly available genome sequences, and experimental confirmation by targeted PCR assays ( Figure S2 and Table S2).
The Bp Core Genome Encodes Essential Processes and a Common Virulence Machinery 86% of the Bp K96243 genes (4619) were found in all strains, representing the Bp core genome ( Figure 1). Using pathway analysis, we found that the core genes were significantly overrepresented in several functions necessary for basic bacterial growth and survival, including amino acid metabolism (1.52610 23 ), inorganic ion transport (3.96610 23 ), nucleotide metabolism (1.52610 22 ) and protein translation (7610 23 ) ( Table 1). The core genes were also significantly enriched in genes conserved in other Burkholderia species (Bp, B. mallei, B. thailandensis and B. cepacia) (p = 8.68610 211 ) (Text S1 and Table  S3)), suggesting that a significant proportion of these Bp core genes may represent core genes in other related species as well [32]. Besides these basic housekeeping functions, the Bp core genes were also significantly enriched in commonly encountered virulencerelated genes such as secretion proteins, capsular polysaccharides, exoproteins, adhesins, fimbriae and pili (p = 1.8610 23 ) (Table 1). For example, three Bp-specific fimbrial gene clusters (BPSL1626-1629, BPSL1799-1801, BPSS0120-0123) were found in all strains. This finding suggests that most, if not all, Bp isolates are likely to possess a common 'virulence machinery'. Notably, many of these conventional virulence genes are also found in other related species such as B. thailandnesis that although non-infectious to

Author Summary
Melioidosis is a serious infectious disease of humans caused by Burkholderia pseudomallei, a soil bacterium endemic to many areas in South East Asia. Besides humans, B. pseudomallei is also capable of infecting many other species and can be isolated from diverse environmental sources including soil, water, and air. In this study, we used DNA microarrays to probe the stability of the B. pseudomallei genome in a large panel of clinical, animal, and environmental strains. We found that evidence of a highly dynamic B. pseudomallei genome, with up to 14% being variably present across different strains. Surprisingly, strains recovered from human patients were significantly associated with the presence of ''genomic islands'', corresponding to regions of DNA directly acquired from other microorganisms. Genes on these genomic islands may thus play an important role in the pathogenesis of human melioidosis. mammals can kill other species such as nematodes [20,33]. This is consistent with the possibility that Bp might have descended from a pathogenic ancestor with a non-mammalian host.

Functional and Chromosomal Biases in the Bp Accessory Genome
14% of the Bp K96243 genome was variable across the strain panel, representing the Bp accessory genome. Since our analysis is confined to genetic elements present in the reference K96243 genome, the extent of genomic variability reported here should be regarded as a lower limit. The 750 variable genes were equally distributed between both Chromosome 1 and Chromosome 2 after normalizing for chromosome size differences. The accessory genes were significantly enriched in paralogous genes (p = 2610 27 ) and genes encoding hypothetical proteins (p = 3610 24 ) ( Table 1). Approximately one-third (30.8%) of the accessory genes were localized to a series of previously identified ''genomic islands'' (GIs) in the K96243 genome [34]. GIs are regions bearing unusual sequence hallmarks, such as atypical GC content and/or dinucleotide frequencies, and are likely to have been recently acquired by lateral gene transfer. Of sixteen GIs in the K96243 genome, fourteen GIs were represented by accessory genes. In contrast, two GIs (7 and 14) were found in all strains, suggesting that GIs 7 and 14 should be regarded as part of the Bp core genome.
Besides the GIs, we also identified several novel regions of at least three contiguous probes that were absent in at least three strains. Henceforth referring to these regions as 'indels', we identified eight indels on chromosome 1, and twelve on chromosome 2 ( Table 2). We experimentally validated two of these indels using PCR assays ( Figure S3). The indels ranged in size from 1.3 to 7.5 kb, and were absent in 12.9% to 45.2% of strains ( Figure 2). Three indels (n1, n4 and n11) were associated with atypical GC content (53.7-58.6%, compared to 68% for the Bp genome), and four (n2, n9, n11 and n16) carried genes characteristic of mobile genetic elements such as integrases, transposases and bacteriophage-related genes, consistent with lateral transfer. These indels may therefore share similar dynamics to the larger genomic islands, and may be considered as genomic ''islets''. In other species, analogous islets which are typically ,10 kb long, have been shown to play a role in virulence (e.g. the sifA islet in S. typhimurium) [35]. Of note, n16 and n18 were flanked at both their 59and 39ends by tandem repeat sequences, while n4, n6, n8 and n19 possessed sequence repeats at either their 59 or 39 ends. In some cases, the islets in the Bp genomes may actually form part of the larger GIs. For example, n2 (BPSL0741-BPSL0744) was located at the 59 boundary of GI 4 (BPSL0745-BPSL0772), while n11 (BPSS0395-BPSS0397) was located immediately 39 to GI 13 (BPSS0378-BPSS0391A).
Three indel regions (n6, n12 and n19) contained genes associated with LPS metabolism. Lipolysaccharides (LPS) are macromolecular components on the outer membranes of Gramnegative bacteria composed of lipid A, core oligosaccharide, and O-antigen polysaccharides [36]. LPS molecules are commonly immunogenic and have been previously implicated in virulence for numerous microbes [37,38]. Region n6 (BPSL2666-BPSL2668) contains a phosphoglucomutase (BPSL2666), a lipopolysaccharide LPS biosynthesis protein (BPSL2667) and a glycosyltransferase (BPSL2668), and was located four genes away from a larger LPS biosynthesis cluster (BPSL2672-BPSL2688). Both regions n12 (BPSS0427 -BPSS0429) and n19 (BPSS2245-BPSS2255) contained two O-antigen related genes, including O-acetyltransferase and glycosyltransferase. While n12 corresponds to a previously identified type III O-PS polysaccharide gene cluster [39], the contribution of n19 genes to Bp LPS biology is currently unknown. The identification of three physically unlinked indels related to LPS metabolism provides a mechanism by which high levels of LPS diversity may be maintained in the Bp population [40].

Unsupervised Clustering Using the Accessory Genome Distinguishes Clinical Isolates from Animal and Environmental Strains
To explore if differences in accessory genome content might be associated with host adaptation or the propensity to cause disease, we applied unsupervised clustering to cluster the strains using the entire set of 750 accessory genes (''accessory genome clustering'', AGC). We identified three large AGC clusters each containing 27 to 42 strains, with each cluster containing at least 4-6 subbranches ( Figure 3). Most strikingly, the majority of human clinical isolates (73.1%) fell into one AGC cluster (Clade C), another cluster contained 73.7% of the animal isolates (Clade A), and a third cluster contained 45% of the environmental isolates (Clade E). Similar results were obtained when the clustering was repeated using either Chromosome 1 or Chromosome 2 accessory genes ( Figure S4). The over-representation of human clinical isolates in the C clade was highly significant (P = 2.001610 214 , Fisher's exact test), and of the remaining 13 clinical isolates nine segregated within the E clade and four in the A clade. This clustering pattern is unlikely to represent differences in geographical distribution, since the majority of the clinical (65%), animal (89%) and environmental isolates (80%) were isolated in Singapore within a ,700 km 2 region or from nearby islands. Furthermore, clinical isolates from Thailand clustered with the other clinical isolates, despite being geographically remote. This analysis therefore suggests that strains associated with human melioidosis may possess an accessory genome distinct from most animal and environmental strains. We also note that all three clades contained environmental isolates, which is consistent with the view that the environment represents a diverse reservoir from which human and animal adapted strains emerge. Clinical Isolates are Associated with the Presence of Genomic Islands We then performed a supervised analysis to identify which of the 750 accessory genes were significantly different between the C and A/E clades. Of the 750 genes, 218 genes were commonly present in isolates in the C clade but absent from strains in the other two clusters ( Figure 4A). Strikingly, we found that almost all of these 218 genes (85%) were localized to the GIs, with all fourteen GIs being represented. This figure (85%) is significantly higher than the 31% of all accessory genes located on GIs, raising the possibility that GIs may play an important role in determining ecological niche and host adaptation.
Is there any direct evidence that genes encoded on GIs, and which define the C clade, might play an important role in the biology or pathogenicity of Bp? Unfortunately, almost 35% of the GI genes encode 'hypothetical' proteins (Table S4), meaning that their function is unknown. For those genes specific to the C clade where functions could be assigned, several broad functional classes were represented. For example, GI8 contains several genes spermidine/putrescine transport genes (potB, potC, potG), which have been associated with biofilm formation and the regulation of Type III secretion genes [41,42]. Type I restriction-modification enzymes are found on GI5 and GI10, and a glutathione Stransferase gene (BPSS2048) on GI16 may impart resistance to oxidative stress. Also supporting their potential role in Bp biology, several GI genes exhibited distinct and complex gene expression patterns during Bp growth (Text S2). However, the role of such genes in pathogenesis remains speculative. In order to explore this further, we generated an experimentally mutated strain (ATS2053) disrupted in BPSS2053, a GI 16 gene encoding a hemagglutininrelated protein, and determined the adherence of the mutant strain to human buccal epithelial cells. A highly significant reduction in the adherence to buccal epithelial cells was noted between the 1026b clinical isolate and the isogenic ATS2053 mutant strain (mean adherence: 1026b -16.363.2 vs ATS 2053 -4.461.7, p,0.001, Students t test). This finding provides evidence pointing both to the biological relevance of GI genes, but more specifically to a role of these genes in virulence.

Comparison of Clustering Patterns Using MLST or the Accessory Genome
Finally, we examined the concordance between strain clusters defined on the basis of accessory gene content and the phylogenetic signal within the Bp core genome. We characterised 45 representative isolates by Multilocus Sequence Typing (MLST), a typing scheme that indexes variation at seven core housekeeping genes [43]. Using the previously published Bp scheme [44], we resolved the 45 isolates into 9 sequence types (ST 46, 51, 54, 84, 169, 289, 414, 422 and 423). Seven of these STs (ST51, 54, 84, 46, 169, 289, 414) have been previously observed in Malaysia, Thailand, and Singapore and two (ST422 and 423) are specific to Singapore [44,45].
Previous analyses of MLST for Bp have highlighted the difficulties in building robust phylogenetic trees for this species, owing to a paucity of informative sites in the concatenated data and frequent homologous recombination [46]. We thus favored a categorical approach to comparing the AGC and MLST data by examining the distribution of sequence types across the three clades defined by the AGC data (Table 3). This analysis revealed that the STs are not randomly distributed between the three clusters, indicating some consistency between the MLST and AGC datasets. Most strikingly, of the 20 ST51 isolates, 17 clustered within the animal-associated clade (A), three within the clinical C clade, and none in the environmental E clade. Of the other STs where at least 4 isolates were observed, all four ST422 isolates corresponded to the C clade, and all four ST84 isolates clustered within the E clade. Finally, of the nine ST423 isolates, five clustered within the C clade and four in the E clade.
These data suggest that the animal-associated clade is likely to correspond to a single clone (ST51) and provides some evidence for concordance between STs 422 and 84 with the AGC data, although the evidence in these latter cases is equivocal due to the small number of strains. In contrast, the ''split'' of the ST423 isolates between the clinical and environmental clades, and the 3 ST51 isolates belonging to the clinical clade, represent clear discrepancies between the two datasets. Possible explanations for these discrepancies may represent convergence of either the MLST or the AGC data, as discussed below.

Discussion
In this report, we present a comprehensive aCGH analysis for a large series of natural Bp isolates. We found that the accessory (variably present) portion of the Bp genome corresponds to ,14% of the whole genome content, which is broadly similar to other cproteobacteria. Since this approach is limited to the detection of elements present in the Bp K96243 genome, and novel elements in query genomes are not detected, this estimated fraction of the accessory genome should be regarded as a lower bound.
In the only published study of a Bp genome sequence to date, Holden et al (2004) computationally identified 16 GIs comprising 6% of the K96243 genome [34], and our data confirm that most of these islands are indeed highly variable between strains. However, two GIs (7 and 14) were found in all strains and should thus be regarded as part of the Bp core genome. Furthermore, our data also revealed the variable presence of several other small genomic islets/indels across the two chromosomes, which might contribute to the phenotypic diversity of Bp. Notably, we observed that several indels (n6, n12 and n19) were related to LPS biology. Currently, the exact contribution of LPS to Bp virulence is unclear. For example, DeShazer et al (1998) showed that Bp type II O-PS is essential for serum resistance and virulence [47], and mice pre-immunized with Bp LPS displayed enhanced survival to a subsequent challenge [48]. In contrast, other groups have reported that Bp LPS exhibits a reduced ability to activate immune cells compared to E. coli LPS, suggesting that LPS might play only a minimal role in Bp virulence. It is possible that these conflicting results might reflect heterogeneity in LPS pathways resulting from the variable presence of these indels, and represent an important mechanism for host adaptation. Interestingly, while it was recently shown that type III O-PS mutants (indel n12) do not appear to exhibit significant virulence attenuation in mouse infection assays [39], we have found in preliminary work that Bp strains lacking the indel n19 LPS cluster generally exhibited lower levels of virulence compared to strains where this cluster was present (SSH, data not shown). In the AGC tree, n19 was absent both from three strains segregating as a single branch in the A clade, and from 5 strains in the C clade that segregated across multiple branches. This suggests that n19 may have been recurrently lost in different Bp lineages. Further experiments are clearly required to understand the role of these LPS clusters in Bp virulence.
We also found that the Bp strains could be clustered into distinct clades based on both the presence and absence of specific accessory genes. Of primary interest, strains belonging to the C clade of clinical isolates were largely defined by the presence of 218 genes, of which 85% are localized to the GIs. These findings provide evidence for a distinct repertoire of Bp genes that may cause a predisposition to human disease and that these genes tend to be located on GIs. Although many of the genes encoded on the GIs are of unknown function, we present experimental evidence that a strain mutated in one of these genes exhibited decreased adherence to human buccal endothelial cells, supporting a role in virulence potential. We also observed coordinated growthassociated expression of several GI genes, which is also consistent with the view that they play an important biological role. What might this biological role be? At present, we consider it most likely that this ''virulent'' combination of genes has likely emerged for reasons other than to cause human disease, particularly since cases of human (or animal) infection are relatively rare compared to the density of Bp in the soil. In contrast to bacteria which are obligately associated with eukaryotic hosts, soil bacteria such as Bp commonly face extreme and unpredictable biotic and abiotic challenges including extreme temperature shifts, solar radiation, variable humidity, competition for nutrients, and the requirement to survive ingestion by predatory protozoa, nematodes, the production of bacteriocides from other bacteria and phage infection. It thus seems entirely plausible that genes facilitating survival against these environmental challenges might have also indirectly enhanced the microbe's ability to colonize and ''accidently'' infect a human host, particularly when the host is immunocompromised [49].
Another possibility that might explain the enrichment of GIs in the clinical isolates is that Bp is undergoing cryptic cycling through normal human hosts (as opposed to the immunodeficient host), and that these GIs are selected during this host-pathogen interaction. In melioidosis-endemic NE Thailand, the majority of healthy individuals have antibodies to Bp by the age of 4 years, indicating a constant exposure to the bacterium that may occur by inoculation, inhalation or ingestion [50]. Within these normal hosts, Bp is likely to spend a period of time being exposed to the effects of the host immune response, after which the microbe may experience bacterial death, persistence, or expulsion from the host in a viable state and subsequent return to the environment. This latter process might occur through skin desquamation or urine and stool, since human excrement commonly finds its way back to the environment. Such cryptic cycling of Bp through the normal human host population could also lead to the selection of factors that promote survival in vivo. However, as we consider the human host to be a relatively minor component of Bp ecology, we argue that this scenario is, on balance, less likely.
The availability of both MLST and aCGH data for a representative sub-sample of isolates also provided us the opportunity to compare clade distributions defined either by accessory genome content or allelic variation in the core genome. We found that the animal associated strains largely corresponded to a single MLST clone (ST51). These isolates were assembled from three distinct sources: the Singapore zoo, the University of Malaya and a pig abbatoir in Singapore. The soil isolates corresponding to ST51 (which also clustered in the A clade) were not isolated from soil samples in proximity to the animal ST51 isolates, which suggests that this genotype is also present in the environment. The homogeneity of these isolates is therefore striking and cannot be explained simply by sampling bias. The consistency between the microarray and MLST data strongly suggest that this clade is monophyletic, and that the strains harbour similar gene repertoires by virtue of common descent.
In contrast, we also observed clear discrepancies between the MLST and aCGH clades. For example, three ST51 isolates  clustered within the clinical aCGH clade, and ST423 was split between the clinical and environmental aCGH clades. There are three possibilities to explain these discrepencies: i) The MLST data represents the ancestral state which is inherited by descent into two AGC-defined clades -this is unlikely for the animal cluster as the vast majority of isolates are ST51, but might conceivably explain the ST423 split between the clinical and environmental clades. ii) Convergence of the MLST alleles -this would imply that isolates with the same ST are not identical by descent but happen to share the same combination of alleles. The presence of a few very common alleles for each gene, combined with high rates of recombination in Bp make this possibility more likely. iii) Independent convergence of gene content to one of the three clusters. Unless large numbers of genes can be transferred in single events, this possibility seems less parsimonious than (ii). More data are required to examine which of these hypotheses is most likely. In summary, our study provides direct experimental confirmation that the Bp genome is highly plastic, and that gene acquisition and deletion are major drivers of this variability. This variability is far from random, and is functionally biased towards genes involved in mobile elements, hypothetical and paralogous genes, and LPS biosynthesis. Furthermore, genes on mobile elements may predispose individual strains, either directly or indirectly, towards causing human disease. We believe this latter result is significant in that most Bp research to date has focused on virulence components in the Bp core genome rather than genes on mobile elements. We conclude by noting that most of the Bp genome sequences currently available have been obtained from human clinical isolates. Given our results, it might be highly informative to subject a panel of animal and environmental Bp isolates to similar detailed genome analysis as well.

Bacterial Strains
Ninety-four Bp isolates were used in this study. These include: a) the K96243 reference strain, b) 52 clinical isolates from melioidosis patients between 1996 and 2005, c) 19 animal isolates from various species (eg monkeys, pigs, birds, and dogs) diagnosed with melioidosis between 1996 and 2000, d) 20 soil isolates from 1994 to 2003, and e) two type strains (ATCC23343 and ATCC15682). All strains were isolated in Singapore, neighboring islands, or surrounding countries (Malaysia, Thailand). The isolates were sampled from a diversity of locations and not a single site, supporting their unbiased nature (Aw Lay Tin and Joseph Tong, personal communication). Further strain information is provided in Table S1.

Genomic DNA Extraction and Array-Based Comparative Genomic Hybridization (aCGH)
Strains were cultured on Tryptone Soy Agar (TSA) (Difco Laboratories, Detroit, Michigan) at 37uC, and genomic DNA extracted using a genomic DNA purification kit (Qiagen). The Bp DNA microarray has been previously described [29][30][31] and comprises approximately 16,000 PCR-amplified array probes representing all 5742 predicted genes in the K96243 genome printed in duplicate. Test genomic DNA (2 mg) was fluorescently labeled with Cy3-dCTP (Amersham Pharmacia Biotech) using nick-translation and co-hybridized to the array with an equal quantity of Cy5-dCTP (Amersham Pharmacia Biotech) labeled reference K96243 DNA. The absence of significant dye-bias artifacts was confirmed by analyzing reciprocal dye-swap hybridizations for 10 isolates data not shown, also see ref [29]. Raw fluorescence data was acquired using an Axon scanner with GENEPIX v4.0 software (Axon Instruments, Redwood City, CA).

Microarray Data Preprocessing
Individual arrays were internally normalized between the Cy3 and Cy5 channels by LOWESS normalization, and the entire dataset was cross-normalized by median-scaling each array to the same Cy3/Cy5 ratio. To filter the microarray data, we eliminated probes exhibiting a missing value score across .40% of samples (indicating that they were not reliably measured), and probes whose genomic loci were redundant with other probes. This data filtering procedure generated a final high-quality data set of 5369 non-redundant probes. The entire microarray data set is available at the Gene Expression Omnibus database under accession number GSE9491.

Identification of Accessory Genes
A Gaussian mixture model (GMM) [51] was used to identify accessory and core genes in the data set. In concept, a GMM fits a test signal distribution (such as microarray data) to either a single or double gaussian curve, and the likelihood that the distribution corresponds to a single curve is computed. The GMM was applied in two stages. First, p-values were computed using the aCGH profiles of each individual array spot, following a chi-square distribution with 3 degrees of freedom under the null hypothesis that the data distribution of the spot follows a 1-gaussian distribution. Second, since each probe was spotted twice on the array, we obtained composite p-values of each array probe using Inverse Chi-square Meta-Analysis [52], squaring the p-values of both spots belonging to the same probe. This latter statistic follows a chi-square distribution with 4 degrees of freedom. All p-values were corrected for multiplehypothesis testing according to the Benjamini-Hocheberg procedure [53]. A cut-off of p#1.83E-08 was selected to define the top 750 most highly variable probes, representing the accessory genome.

Pathway Analysis of Core and Accessory genes
All protein coding sequences in the Bp K96243 genome were queried by BLASTP against the Cluster of Orthologous group (COGs) database, a public bioinformatic database that groups protein sequences on the basis of phylogenetic similarity to various cellular functions, such as protein translation, DNA replication and transcription, nuclear structure and defense mechanisms (accessible at http://www.ncbi.nlm.nih.gov/COG/new/). Matches were defined as database hits with an e-value threshold of ,10 26 . Based on the COG assignments, the K96243 proteins were assigned to functional categories. Fisher's exact tests were used to identify significantly overrepresented COG categories in either the core or accessory genes. To identify conserved genes (metagenes) across four Burkholderia species, we queried the 3460 Chr 1 and 2395 Chr 2 ORFs in the Bp K96243 genome against the B. cenocepacia (Bc), B. mallei (Bm), and B. thailandensis (Bt) genomes using tblastn [32] (Text S1). To minimize the number of ambiguous predictions including ORFs with matches to multiple genomic locations, we constrained the resulting matches to have I) a minimum length of 50 amino acids, II) a minimal e-value cut-off of 1e-6 and III) a minimum percent identity of 50%. Homology assignments returned 2675 genes and were validated by a reciprocal blast assay resulting in 2590 genes. Control analyses using either Bc, Bm or Bt as starting reference genomes yielded similar metagene sets (data not shown). Paralogous genes were identified using the CD-HIT program [54] as genes with .60% identity to one another, following established studies [55,56]. Tandem repeat regions in the K96243 genome were identified using the Tandem Repeats Finder program [57].

Clustering Analysis
Phylogenetic trees based on aCGH profiles were constructed using MultiExperiment Viewer (MeV) version 4 (http://www.tm4. org/mev.html) using an average linkage clustering algorithm with a Euclidean distance metric. Support trees were based on 1000 bootstrap samples. Neighbor-joining trees based on MLST sequence data were constructed by MEGA ver. 2.1 software using the Kimura-2-parameter method of distance estimation. eBURST v3 (http://eburst.mlst.net) was used to demonstrate relationships between closely related STs (those differing at only a single locus) [58,59], with the tree files visualized using PhyloDraw [60].

Construction of Mutants
The BPSS2053 (fhaB) gene was disrupted in strain DD503, an isogenic derivative of wild-type 1026b. In DD503, the amr locus, encoding a multidrug efflux system, has been experimentally deleted [61]. The increased antibiotic susceptibility of DD503 makes it a useful strain for allelic exchange experiments as it allows the use of currently available allelic exchange vectors. There is no significant difference in virulence between the1026b parent strain and DD503 [61]. A 1036-bp internal region of the BPSS2053 (fhaB) gene was amplified by PCR using primers 53F:TGGTGGTGCAAGA-GAATGGC and 53R:ATCGTGACCGATTGCTTGCC from Bp 1026b chromosomal DNA as previously described [21]. The PCR product was cloned into pCR2.1-TOPO (Invitrogen Life Technologies, Burlington, Ontario, Canada) according to the manufacturer's instructions. The internal region from BPSS2053 was cloned as an EcoR1 fragment into pGSV3-lux, a suicide vector containing a promoterless lux operon as a reporter, to create pATS2053. The recombinant plasmid pATS2053 was transformed into E. coli SM10lpir [62]. Transformed E. coli containing pATS2053 were conjugated with Bp DD503, and transconjugants selected on LB-gentamicin-polymyxin B agar. The transconjugants were screened for lux-mediated light production by assaying 100 ml of overnight broth cultures of individual colonies. One of the lightproducing transconjugant strains was designated as Bp ATS2053.

Adherence Assays
Adherence of BPSS2053 (fhaB) mutants (Bp ATS2053) to human buccal epithelial cells in vitro were compared against wild-type parental Bp 1026b as previously described [63]. Briefly, buccal epithelial cells from healthy control individuals were isolated by vigorous scraping of the buccal mucosa with a cotton-tipped swab. The swabs were placed into phosphate buffered saline (PBS), transported to the laboratory, and the epithelial cells were incubated in vitro with bacteria at a ratio of 100 bacteria to 1 epithelial cell for 1 h at 37C in a shaking water bath. Unattached bacteria were removed from the mixture by repeated washing with PBS and centrifugation. Bacteria per cell were counted following staining of the bacteria-cell mixture with methylene blue by counting the number of bacteria attached to each of 50 cells and obtaining a mean number of bacteria/cell.

Multilocus Sequence Typing (MLST)
MLST on 45 strains was performed as described in Godoy et al (2003) [44] using primer pairs for seven housekeeping genes (ace, gltB, gmhD, lepA, lipA, narK ndh) on Bp chromosome 1. A complete list of primer pair sequences and PCR conditions is provided in Table S5. Alleles at each of the MLST loci were assigned using the B. pseudomallei MLST website (http://bpseudomallei.mlst.net/)each allele was assigned a different allele number and the allelic profile (string of seven integers) was used to define the sequence type (ST). Sequences that were not in the database were checked by re-sequencing, assigned as new alleles and deposited in the MLST allele database.


extfieldsofstudy: Chemistry Medicine

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Study of a Synthetic Human Olfactory Receptor 17-4: Expression and Purification from an Inducible Mammalian Cell Line

In order to begin to study the structural and functional mechanisms of olfactory receptors, methods for milligram-scale purification are required. Here we demonstrate the production and expression of a synthetically engineered human olfactory receptor hOR17-4 gene in a stable tetracycline-inducible mammalian cell line (HEK293S). The olfactory receptor gene was fabricated from scratch using PCR-based gene-assembly, which facilitated codon optimization and attachment of a 9-residue bovine rhodopsin affinity tag for detection and purification. Induction of adherent cultures with tetracycline together with sodium butyrate led to hOR17-4 expression levels of ∼30 µg per 150 mm tissue culture plate. Fos-choline-based detergents proved highly capable of extracting the receptors, and fos-choline-14 (N-tetradecylphosphocholine) was selected for optimal solubilization and subsequent purification. Analysis by SDS-PAGE revealed both monomeric and dimeric receptor forms, as well as higher MW oligomeric species. A two-step purification method of immunoaffinity and size exclusion chromatography was optimized which enabled 0.13 milligrams of hOR17-4 monomer to be obtained at >90% purity. This high purity of hOR17-4 is not only suitable for secondary structural and functional analyses but also for subsequent crystallization trials. Thus, this system demonstrates the feasibility of purifying milligram quantities of the GPCR membrane protein hOR17-4 for fabrication of olfactory receptor-based bionic sensing device.

Introduction
Membrane proteins are of vital importance to life, as evidenced by the fact that ,30% of the genes in almost all sequenced genomes code for membrane proteins [1][2][3]. However, our understanding of the structures and functions of membrane proteins has lagged behind the known soluble proteins. As of June 2008, there are only 160 unique membrane protein structures known [http://blanco.biomol. uci.edu/Membrane_Proteins_xtal.html], which constitutes less than 1% of all known protein structures. The major bottleneck in obtaining membrane protein structures is the notorious difficulty involved in expressing and purifying the large quantities of membrane protein sample required for X-ray crystallography. In order to accelerate membrane protein structural and function studies, simple and reliable methods for membrane protein production must be developed.
Olfactory receptors (or odorant receptors) are an extremely large class of G-Protein Coupled Receptors (GPCRs) that function together combinatorially to allow discrimination between a wide range of volatile molecules [4,5]. All GPCRs are integral membrane proteins with seven transmembrane domains arranged in a barrel-like conformation. In olfactory receptors, it is believed that this configuration forms a funnel-shaped pocket for odorant recognition [6]. The olfactory receptor (OR) gene family constitutes the largest single class of genes in the vertebrate genome (2-3% in the human). Current estimates put the number of human olfactory receptor genes at 636, with only 339 being functional and the rest being non-functional pseudogenes [7]. This is considerably less than the mouse OR gene family of 1209 (913 functional) [8] or the canine OR gene family of roughly 1200 (,1000 functional) [9]. Despite the fact that they represent the largest class of known membrane proteins, no detailed structure exists for any olfactory receptor and the functional mechanisms of these amazing receptors remains unknown. The crucial first step to enable such pivotal studies is to engineer systems with the capacity to generate and purify milligram quantities of an olfactory receptor.
Mammalian olfactory receptors are expressed on the cilia of olfactory neurons within the nasal cavity. Odorant binding and recognition leads to activation and release of the olfactory G-protein G olf , which triggers cyclic-AMP production, ion-channel-mediated Ca2+ influx, and finally the firing of an action potential into the olfactory bulb to be interpreted by the brain [10]. Through an unknown mechanism of allelic inactivation, every olfactory neuron chooses a single OR gene to express. Signals from neurons that express the same olfactory receptor later converge downstream at neural foci called glomeruli [11]. As the same odorant will stimulate multiple ORs (and to various strengths), the brain receives a spatial map of receptor activity through these glomeruli [12]. Odorants are thought to be recognized by matching a specific spatial pattern (a combinatorial code) [5].
The human olfactory receptor 17-4 (hOR17-4, alternately known as OR1D2) is of particular interest since, in addition to olfactory neurons, it is also expressed on the midpiece of human spermatozoa [13]. Sperm expressing hOR17-4 were found to migrate towards known hOR17-4-responsive odorants such as bourgeonal. Thus the receptor serves a dual role in that it recognizes odorants in the nose as well as plays a potential role in sperm chemotaxis and fertilization. As structural studies of hOR17-4 would not only provide information crucial to understanding the molecular mechanism(s) of olfaction but also have application to human reproduction and population control, we thus selected this receptor as our prototype OR for expression and purification trials.
The GPCR family represents one of the most important known receptor classes as evidenced by the fact that half of all pharmaceutical drugs target GPCRs [14]. Despite their crucial role in mediating such diverse physiological processes as sight, smell, and the response to hormones and neurotransmitters, extremely little is known about these receptors at the structural level. A major breakthrough in 2007 was the determination of only the second GPCR crystal structure-that of a highly engineered human beta2-adrenergic receptor expressed in Sf9 insect cells where intracellular loop 3 was replaced with either antibody or T4 lysozyme to facilitate crystallization [15,16]. Rhodopsin is perhaps the only GPCR that can be easily extracted from tissue, and this may explain why it was the first GPCR to have a detailed structure determined [17][18][19]. However, recent advances in the Khorana laboratory have led to the development of specific mammalian HEK293 cell lines for heterologous expression as well as methods for purification that yield milligram quantities of functional rhodopsin which is suitable for functional analysis and structural study [20][21][22]. We recently used same rho1D4-tag system for a one-step affinity purification of the human tetraspanin membrane protein CD81 [23]. Here we show that this system can be also adapted to facilitate the production and purification of another GPCR, the human olfactory receptor 17-4. In order to carry out biochemical and structural analyses of olfactory receptors as well as engineer olfactory receptor-based biosensor devices, large quantities of receptors are required. Here we here report inducible expression, large-scale production of human olfactory receptor hOR17-4. We bioengineered the synthetic hOR17-4 gene into 50 oligonucleotide fragments, selfassembled them through high cycle PCR and inserted the assembled gene into an inducible human embryonic kidney cell line (HEK293S). We then induced its production using a combination of tetracycline and sodium butyrate. After systematic detergent screening, the zwitterionic detergent fos-choline-14 (FC14) was found to be most effective for solubilization and was subsequently used throughout the entire solubilization and purification. To our knowledge this is the first time an olfactory receptor has been purified from a mammalian cell line.

Construction of synthetic hOR17-4-rho gene
To fabricate synthetic gene constructs we utilized a PCR-based method of gene synthesis [24] that involves parsing the DNA sequence into a set of small overlapping oligonucleotides. During an initial assembly PCR, these oligos function as both primer and template, while the DNA polymerase successively builds longer and longer fragments with each round of PCR. A second amplification PCR then enriches for the full-length gene ( Figure  S1). This process has more recently become known as PCA, or polymerase construction and amplification. To assist in parsing the sequence into oligos ( Figure S2), we used the online program DNAWorks (http://helixweb.nih.gov/dnaworks) developed by Hoover and Lubkowski [25].
The 312 amino acid sequence of wild-type human olfactory receptor hOR17-4 was obtained from GenBank and directly inputted into the software in protein mode. To adapt the resulting gene for use in our specific expression system and to facilitate purification and detection, the reverse-translated DNA sequence was human codon-optimized and appended with a C-terminal rho1D4 epitope tag (TETSQVAPA) [20][21][22]. The synthetic DNA corresponding to the hOR17-4 gene consisted of 1004 bp, encoding a receptor protein of 323 amino acids ( Figure 1). Other than the addition of the rho1D4 tag and linker, the hOR17-4 protein sequence is wild type and completely full length. Noteworthy in the synthesis procedure was the requirement for a high number of PCR cycles (45) during the assembly PCR, presumably due to inefficiencies resulting from non-productive oligonucleotide mispairings.

Induction of hOR17-4 expression in stable HEK293S cell lines
To minimize the toxic effects of receptor overexpression, stable hOR17-4-inducible HEK293S cell lines were created using the Invitrogen T-REx tetracycline regulation system (Materials and Methods). This allowed large-scale cell culture batches to be grown and then, when desired, concerted production of fresh olfactory receptor to be induced in nearly 100% of the cells. Results for the induction of hOR17-4 expression in two of the subcloned HEK293 lines are shown in Figure 2A. Western immunoblotting using a monoclonal antibody against the rho1D4 tag revealed major immunoreactive bands at approximately 32 kD and 60 kD, which correspond in size to monomeric and dimeric forms of the hOR17-4 receptor. This size pattern has been reported previously for several solubilized olfactory receptors expressed in Sf9 and mammalian cells [26][27][28]. Larger molecular weight complexes were also present, presumably due to aggregation and precipitation of the receptor. As sample boiling only increases the precipitation, it is possible that increased temperatures (above 4uC) caused by the electrophoresis could be causing the receptor to partially aggregate. Thus these high MW species could be a side effect of the SDS-PAGE electrophoresis and not originally present in the solubilized receptor fractions.
The histone deacetlyase inhibitor sodium butyrate has been demonstrated to synergistically enhance expression when used with tetracycline-regulated systems [21]. Induction of hOR17-4 in HEK293S cells using tetracycline in conjunction with sodium butyrate increased expression by approximately 4-5 fold over tetracycline alone at all time points tested ( Figure 2B). There was no detectable expression of hOR17-4 in the absence of tetracycline or with sodium butyrate alone. Significant cell toxicity and death was observed in treatments combining tetracycline and sodium butyrate (5 mM) at the 48 and 72 hours time points. Treatment with sodium butyrate or tetracycline alone did not show this toxicity, indicating it to be a result of the high level expression induced by the drugs in conjunction.
To characterize this effect the samples were subjected SDS-PAGE analysis ( Figure 3A). The gel showed two monomer band sizes, approximately 30 kD and 32 kD, suggesting distinct monomer forms (and corresponding dimer forms). It is possible these size discrepancies are due to differences in glycosylation of the receptor. Sodium butyrate addition for 24 hours showed a very large increase in expression over tetracycline alone, with the monomer band running at approximately 32 kD. Sodium butyrate addition for longer periods further increased total expression, however this caused the appearance of the additional monomer form running at 30 kD. The 48-hour time point contained roughly equal parts of both forms while the 72-hour time point consisted predominantly of the 30 kD form. To attempt to avoid the aforementioned toxicity we next performed a time course using a range of sodium butyrate concentrations ( Figure 3B). We discovered that high levels of expression appeared to correlate with the appearance of the 30 kD form (and correspondingly smaller dimer form) and the observed cytotoxicity noted previously.
For subsequent purification experiments, we selected a treatment consisting of sodium butyrate (1 mM) with tetracycline (1 mg/ml) for 48 hours for attempts to purify primarily the 32 kD form. However, to further compare and characterize both forms, increasing the sodium butyrate concentration to 5 mM could be used, as relatively equal amounts of both monomer forms would be present.
We first performed a small-scale purification from six 150 mm culture plates of hOR17-4-inducible HEK293S cells. The plates were treated as to have equal amounts of the 30 kD and 32 kD bands upon harvesting (tetracycline plus 5 mM sodium butyrate, 48 hours after induction). Following a thorough wash procedure to remove non-specific impurities, the bound receptors were eluted by the addition of an excess of epitope peptide (TETSQVAPA). Fractions were subjected to SDS-PAGE followed by either western immunoblotting ( Figure 4A) or total protein staining using highlysensitive SYPRO-Ruby ( Figure 4B). The receptor was completely captured by the bead matrix, as no hOR17-4 was detected in the flow through by western blot. The bound OR eluted primarily in the first and second elution fractions. Total yield of hOR17-4 was approximately 30 mg per 150 mm plate. Mass spectrometry analysis on samples isolated from SDS-PAGE gel bands confirmed the identity of putative monomer and dimer protein bands as hOR17-4 receptor (see Materials and Methods). To expand the system to potential milligram scale, fifty 150 mm culture plates of hOR17-4-inducible HEK293S cells were used. The plates were treated tetracycline plus 5 mM sodium butyrate, 48 hours). The total yield of hOR17-4 following immunoaffinity purification was 1.5 milligrams. To further purify the receptor and to remove the elution peptide, the hOR17-4 was subjected to size exclusion chromatography (SEC) using a gel filtration column on an Ä kta HPLC system. Column flowthrough was monitored by UV absorption (280 nm and 215 nm) and separated into fractions by an auto-fraction collector. As seen in Figure 5A, five distinct peaks were observed. The peak fractions were then pooled, concentrated and subjected to SDS-PAGE followed by total protein staining. As seen in Figure 5B, peak 3 contains monomeric hOR17-4 (.90% purity) while earlier peaks contained largely dimeric (peak 2) and aggregated/oligomerized (peak 1) forms. Peak 4 showed no visible protein and peak 5 corresponds to the residual elution peptide Figure 2. Induction of hOR17-4 in stable HEK293S cell lines. A) Stable inducible cell lines were generated in HEK293S cells using the T-REx system (Invitrogen) expressing hOR17-4 tagged with the rho1D4 tag (TETSQVAPA) at the C-terminus. The pooled cells were then subcloned, expanded, and then tested for induction in media supplemented with (+) or without (2) 1 mg/ml tetracycline for 48 hours. Levels of hOR17-4 were probed via SDS-PAGE western blotting using the rho1D4 antibody. Clones 1 and 5 showed the highest levels of induction while maintaining undetectable background levels in the absence of tetracycline. Clone 5 was selected for all subsequent experiements. B) Addition of sodium butyrate enhances induced expression of hOR17-4. Inducible HEK293 was subjected to a dosage time course using the indicated concentrations of tetracycline and sodium butyrate. Samples were harvested, subjected to dot blot analysis (western blot against rho1D4), and the results quantified by spot densitometry. Tetracycline in conjunction with sodium butyrate increased expression by approximately 4-5 fold over tetracycline alone at all time points tested. Tetracycline was necessary to cause induction, as no expression was detected if sodium butyrate was used alone. Additionally, increasing tetracycline concentration to 2 mg/ml had no significant effect on induction levels. doi:10.1371/journal.pone.0002920.g002  Figure 2) were normalized to total protein content and subjected to SDS-PAGE western blotting against rho1D4. All lanes were treated with 1 mg/ml tetracycline with (+) or without (2) sodium butyrate (5 mM) for the time indicated. B) hOR17-4-inducible HEK293S cells were subjected to a dosage time course using the indicated concentrations of sodium butyrate. All samples were co-treated with 1 mg/ml tetracycline. Samples were normalized to total protein content and subjected to SDS-PAGE western blotting against rho1D4. doi:10.1371/journal.pone.0002920.g003 (TETSQVAPA) from the immunoaffinity purification. The final yield of purified hOR17-4 monomer was 0.13 milligrams (2.6 mg per plate). Thus, using a two-step procedure we have successfully obtained significant amounts of hOR17-4 in a highly pure form.

Discussion
In this study, we successfully developed methods for the construction of inducible mammalian cell lines that generate large quantities of olfactory receptor on demand. To our knowledge this is the first olfactory receptor to be purified from a mammalian cell line. Currently, we have demonstrated the production of the human olfactory receptor hOR17-4 in a stable tetracyclineinducible human embryonic kidney cell line (HEK293S). Expressed OR genes were fabricated from scratch using PCR-based gene synthesis (polymerase construction and amplification, aka PCA), which facilitated codon optimization for high level expression and attachment of affinity tags for detection and purification. The HEK293S cells can be grown and OR expression induced in adherent cultures (yield of ,30 micrograms/150-mm plate). Using methods originally adapted from the production and purification of the bovine GPCR rhodopsin [20][21][22] and further optimized (including the full-spectrum screening of over 70 detergents), the olfactory receptor is solubilized and extracted from the cells using fos-choline-14. The OR protein is then isolated using a two-step purification method (immunoaffinity followed by size-exclusion chromatography) which yields hOR17-4 monomer at greater than 90% purity ( Figure 5B).
There have been a host of previous studies that have expressed and studied olfactory receptors in native and heterologous systems. . Immunoaffinity purification of hOR17-4. Six 150 mm tissue culture plates of were grown to 90% confluence then induced and treated with tetracycline (1 mg/ml) and sodium butyrate (5 mM) for 48 hours. Plates were scrape harvested, solubilized, and the processed lysate subjected to immunoaffinity purification using rho1D4 antibody linked to sepharose beads for capture. Bound proteins were washed and then eluted using the nonapeptide TETSQVAPA. Samples were subjected to SDS-PAGE followed by either western immunoblotting with rho1D4 antibody (A) or total protein staining with Sypro Ruby (B). Black triangles indicate the 30 and 32 kD monomer forms. Tot, total lysate; FT, flow through; W, wash; E, elution. doi:10.1371/journal.pone.0002920.g004 However, to date there has not been a case where olfactory receptors have been overexpressed and purified to homogeneity in significant quantities. While purification of ORs has been attempted in bacterial [33] and Sf9 insect systems [26][27], these were unable to produce large quantities of native full-length olfactory receptor. Additionally, these organisms lack mammalian post-translational machinery and thus purified receptors may be improperly folded or missing critical modifications. It is known that most, if not all, GPCRs are glycosylated, and indeed the olfactory receptors have conserved Nlinked glycosylation sites (Asn-X-Ser/Thr) at their N-terminus [34][35]. Several studies have indicated that loss of glycosylation can lead to improper folding and targeting, resulting in decreased function and compromised structure [36][37]. Loss of either N-terminal glycosylation site (Asn-2 or Asn-15) of rhodopsin is sufficient to cause loss of signal transduction despite no apparent change in localization or folding [38][39]. This indicates that purification of olfactory receptors from mammalian systems might be crucial for functional expression and purification.

Characterization of purified hOR17-4
It is interesting to note that the engineered hOR17-4-rho protein (with theoretical molecular mass of 36.2 kD) migrates slightly faster than expected on SDS-PAGE gels (30 kD and 32 kD). One possible explanation for this discrepancy is incomplete receptor denaturation by SDS since boiling the samples results in aggregation. Indeed, many other membrane proteins have been found to migrate faster than their actual size on SDS-PAGE, and this has been reported for other olfactory receptors as well [27,33]. We believe both monomer forms of the expressed hOR17-4 receptor to be intact, full-length proteins as evidenced by: 1) detection by the C-terminal rho1D4 monoclonal antibody, 2) detection by custom anti-hOR17-4 polyclonal antibodies raised against the hOR17-4 N-terminus or C-terminus, 3) detection of a 30 kD N-terminally 6xHis-tagged hOR17-4 variant using anti-His antibodies (data not shown). Additionally, we see only the 32 kD form at low levels of induction while the 30 kD form only begins to appear at higher levels of induction. One explanation is that the receptor is glycosylated at low induction but that the toxicity associated with higher expression causes the receptor to begin to accumulate in the ER and not be properly processed.
The appearance of two distinct hOR17-4 monomer bands following purification could pose a problem for structural studies using X-ray crystallography, since typically a high degree of protein homogeneity is required for protein crystallization. We initially believed that it was possible to obtain primarily 32 kD form using lower levels of induction ( Figure 3B). However, we have observed that the rho1D4 immunoaffinity purification appears to increase the proportion of 30 kD monomer form relative to 32 kD form given the original treatment. For example, the treatment dosages for the small-scale ( Figure 4) and large-scale ( Figure 5) purifications were chosen (based on data in Figure 3B) to result in equal amounts of both forms or primarily 32 kD form, respectively. However, following immunoaffinity purification the resulting elution samples were enriched in the 30 kD monomer form. One hypothesis is that the 30 kD (potentially nonglycosylated form) binds more readily to the rho1D4-coupled bead matrix. Therefore, to obtain truly homogeneous hOR17-4 monomer it might be necessary to use even lower levels of induction where no 30 kD form exists. Another option would be to mutate the asparagine in the consensus N-glycosylation site of the receptor, located at amino acid position 5. We predict such a change to result in exclusive production of the 30 kD monomer form. However, the functional effect of abolishing glycosylation on the receptor is unknown.
There have been numerous reports in the literature citing difficulties in expressing functional olfactory receptors in heterologous systems. The main problem seems to be improper membrane targeting and resulting cytosolic localization of the majority of ORs [35][36][40][41]. Several studies have been able to alleviate this problem by the addition of putative membrane import signals to the N-terminus of the olfactory receptor [42][43]. However, more recent studies [44][45] have expressed functional olfactory receptors in HEK293 cells without the use of such signal tags. We also investigated the use of an N-terminal ''membrane import'' sequence composed of the first twenty amino acids of bovine rhodopsin but found no significant increase in receptor yield or localization (results not shown).
Our largest adherent culture experiment consisted of fifty 150 mm tissue culture plates. While the total yield of crude hOR17-4 following immunoaffinity purification was 1.5 milligrams, the subsequent size exclusion chromatography and associated concentration steps reduced this yield to 0.13 milligrams of purified hOR17-4 monomer (2.6 mg per plate). The monomer was .90% pure, with the only other major contaminating band consisting of hOR17-4 dimer. While this represents a significant milestone, using adherent culture for milligram-scale purification of the receptor monomer poses a substantial challenge. However, the HEK293S cell line is capable of suspension culture, and we plan to scale-up purification yields by adapting the system to culture in a large volume (5-10 liter) liquid bioreactor. The high cell densities allowed should allow the production and subsequent purification of milligram quantities of olfactory receptors, which will be necessary for future experiments in determining receptor structure and function.

General application for the purification of other olfactory receptors
Here we show that a GPCR olfactory receptor gene can be designed, synthesized, placed into an inducible mammalian expression system and the resulting full-length protein purified to near homogeneity in a two-step process. In addition to our experiments on hOR17-4, we have constructed optimized synthetic genes for several mouse olfactory receptors and initial expression trials have proven successful. The small size of the rho1D4 tag and its extremely mild elution conditions provide a minimum of disruption to the purified protein. In contrast to previous attempts at OR purification using bacterial systems which required fusion to GST and truncated or mutated OR protein sequences [33], the system described here allows for the production of full-length wild-type OR. The application of this technique to other olfactory receptors could feasibly lead to a generalized method for obtaining large quantities of any olfactory receptor in a rapid and simple manner. Such methods could prove extremely useful in elucidating the structural and functional mechanism(s) of olfactory receptors and in their integration into OR-based bionic sensing devices.

Simple and adaptable methods
Membrane proteins are natural nanobiodevices; they perform exquisitely fine functions, from specific ion channels, chemical transporters, signal transduction, and information processing, to photosynthesis for direct solar energy harvesting. In order to accelerate determination of membrane protein structural studies, simple and reliable methods are necessary to attract more and more researchers to study these elusive membrane proteins and use them to fabricate new bionic devices. We here show that the GPCR olfactory receptor gene can be bioengineered, placed into an inducible system and to purify to near homogeneity in a straightforward 2-step process. Our effort to purify hOR17-4 is a very important step not only for us to understand the secret of smell, but also to design new bionic nose sensing devices.

Gene Construction
The protein sequence for hOR17-4 (also known as OR1D2) was obtained from GenBank (NCBI Accession # NP002539). DNA-Works online software (http://helixweb.nih.gov/dnaworks) was used in protein mode to design the synthetic gene and parse it into an oligonucleotide set. To adapt the synthetic hOR17-4 olfactory receptor gene for use in mammalian cell expression and purification, the following sequence modifications were made: i) human codon optimization; ii) addition of a C-terminal rho1D4 epitope tag (TETSQVAPA) preceded by a two glycine linker to facilitate detection and purification; iii) addition of a Kozak consensus sequence (GCCACCACC) immediately 59 to the ATG start codon; iv) addition of an EcoRI restriction site at the 59 end and a NotI restriction site at 39 end of the gene to enable cloning into expression vectors. The synthetic hOR17-4 gene consisted of 1004 bp, of which 969 bp code for the 323 amino acid hOR17-4-GlyGly-rho1D4 protein. The designed oligonucleotide primers were purchased from IDT (Coralville, IA) with a maximum length of 45 bp ( Figure S2). PCR-based gene synthesis was performed using a 2-step assembly/amplification protocol ( Figure S1) [46] with the exception that the assembly PCR was run for 45 cycles. PCR reactions were then analyzed by gel electrophoresis and stained with ethidium bromide. Full-length product was excised, extracted, and then digested with the pertinent restriction enzymes. The genes were then ligated into the T-REx pcDNA4/To inducible expression plasmid (Invitrogen, Carlsbad, CA), sequenced, and a correct clones grown up using a MaxiPrep kit (Qiagen, Valencia, CA). The plasmid containing the optimized hOR17-4 gene was designated pcDNA4/To-hOR17-4-rho1D4.

Generation of Stable Inducible Cell Lines
HEK293S (suspension adapted HEK293 cells) containing the stable expression of pcDNA6/Tr (Invitrogen) which encodes the Tet repressor protein (TetR) had previously been generated and cloned [21]. HEK293S cell monolayers were grown in DMEM/ F12 with GlutaMAX (Invitrogen catalog # 10565-042) supplemented with fetal bovine serum (10%), HEPES (15 mM), nonessential amino acids (0.1 mM), sodium pyruvate (0.5 mM), penicillin (100 units/ml), streptomycin (100 mg/ml) and grown at 37uC at 5% CO 2 . All tissue culture and media components were purchased from Invitrogen unless otherwise noted. The pcDNA4/ To-hOR17-4-rho1D4 plasmid was then transfected into these cells using Lipofectamine 2000 and after 48 hours cells were subjected to drug selection in 5 mg/ml blasticidin and 250 mg/ml zeocin for 2 weeks and then subcloned. 28 colonies were expanded and screened for inducible expression using media supplemented with or without 1 mg/ml tetracycline for 48 hours. Samples were then scrape harvested, solubilized in phosphate buffered saline (PBS) with 2% w/v Fos-Choline-14 (Anatrace, Maumee, OH) and Complete Protease Inhibitor Cocktail (Roche, Basel, CH) for 1 hour at 4uC. Expression was assayed via dot blotting and SDS-PAGE western blotting using the mouse monoclonal antibody rho1D4. Clone 5, the colony showing the best expression of hOR17-4 under induction conditions while maintaining undetectable expression without induction, was selected and expanded into large-scale culture and used for all subsequent experiments. The hOR17-4-inducible HEK293S cell line was maintained using 5 mg/ml blasticidin and 250 mg/ml zeocin.
For initial dosage and time course experiments, hOR17-4inducible HEK293S cells were grown to 80-90% confluency at 37uC in 6-well tissue culture plates, treated as indicated and then scrape harvested into ice-cold PBS containing Complete Protease Inhibitor Cocktail. The hOR17-4 was then solubilized by resuspending the cell pellets in 150 ml solubilization buffer and rotating for 1 hour at 4uC. The non-solubilized fraction was then pelleted using at 13,000 g for 30 minutes. The supernatant was then removed and analyzed by SDS-PAGE.
For purification experiments, up to fifty 150 mm tissue culture plates were used per experiment. Briefly, hOR17-4-inducible HEK293S cells were seeded at a density of 5610 6 cells per 150 mm dish and grown for 72 hours at 37uC, at which point they reached 80-90% confluency. The cells were then induced with medium containing tetracycline (1 mg/ml) plus sodium butyrate (as indicated). After 48 hours, the cells were harvested by scraping (at 4uC) each plate into 2 ml PBS containing Complete Protease Inhibitor Cocktail. The cells were then pooled and snap frozen in liquid nitrogen and stored at 280uC until purification was carried out. On the day of purification, cells were thawed on wet ice and spun down by centrifugation at 4000 g for 1 minute. All further steps were performed at 4uC unless noted. The hOR17-4 was then solubilized by resuspending the cells in solubilization buffer (1-2 ml per 150 mm plate) and rotating for 4 hours. The non-solubilized fraction was then pelleted using an ultracentrifuge at .100,000 g for 30 minutes. The resulting supernatant was removed and put at 4uC. A small amount of supernatant (100 ml) was set aside, labeled ''total lysate'' and stored at 220uC. The remainder was directly applied to immunoaffinity purification.

Immunoblotting and Total Protein Staining
Samples were assayed via polyacrylamide gel electrophoresis (SDS-PAGE) under both reducing and denaturing conditions. Samples were prepared and loaded according to standard Novex gel protocols with the exception that the samples were incubated at room temperature prior to loading, as boiling caused membrane protein aggregation. Full Range Rainbow (GE Healthcare, Waukesha, WI) molecular weight marker was loaded as the protein size standard. Samples were resolved on Novex 10% Bis-Tris SDS-PAGE gels (Invitrogen) were run using NuPAGE MOPS buffer at 100 V and were subsequently transferred to a 0.45 mm nitrocellulose membrane and subjected to western immunoblotting using the rho1D4 as primary antibody, followed by a secondary HRP-linked goat anti-mouse IgG (Pierce, Rockford, IL) and detection using the ECL-Plus Kit (GE Healthcare). For total protein staining, SDS-PAGE gels were run as above, stained using SYPRO-Ruby (a more sensitive alternative to Coomassie; Invitrogen), and visualized by fluorescence using UV transillumination (excitation wavelength 300 nm). All western blot and SYPRO-Ruby images were captured using a Fluor Chem gel documentation system (Alpha Innotech, San Leandro, CA).

Immunoaffinity Purification
For immunoaffinity purification we utilized rho1D4 monoclonal antibody (Cell Essentials, Cambridge, MA) chemically linked to CNBr-activated Sepharose 4B beads (GE Healthcare). The rho1D4 elution peptide Ac-TETSQVAPA-CONH 2 was synthesized by CBC Scientific (San Jose, CA). Rho1D4-sepharose immunoaffinity purification has been described previously [21]. Briefly, the cell extract supernatant was mixed with rho1D4-coupled sepharose bead slurry (binding capacity 0.7 mg/ml) and rotated overnight at 4uC to capture the rho1D4-tagged olfactory receptors. The beads were then pelleted by centrifugation at 2000 g for one minute and the supernatant collected, labeled as ''flow-through'' and saved for future analysis. The beads were then re-suspended in 100 bead volumes of cold wash buffer (PBS+0.2% Fos-Choline-14), rotated for 10 minutes at 4uC, then re-pelleted. A total of five washes were carried out and 100 ml of each sequential wash was saved for subsequent analysis. After the final wash, the beads were pelleted again and transferred to a new tube for elution. A series of five elutions (each rotated 1 hour at room temperature) was then carried out, each using 1 bead volume of elution buffer (PBS+0.2% FC14+100 mM TETSQVAPA peptide). Total protein concentration was measured using BCA assay (Pierce).

Mass Spectrometry
Immunoaffinity-purified samples of hOR17-4 were separated via SDS-PAGE, stained with SYPRO-Ruby and gel bands at 30 kD, 32 kD, and 60 kD were excised into sterile, methanolrinsed microcentrifuge tubes. The samples subjected to trypsin digestion and the resulting fragments analyzed by Ion Trap LCMS for protein identification by the MIT Biopolymers Laboratory (Cambridge, MA). All bands were identified as hOR17-4, indicating monomeric and dimeric forms.

Size Exclusion Chromatography
For further purification, hOR17-4 proteins were subjected to gel filtration chromatography using a HiLoad 16/60 Superdex 200 column on an Ä kta Purifier HPLC system (GE Healthcare). The column was first equilibrated using wash buffer (PBS+0.2% w/v Fos-Choline-14). Pooled hOR17-4 elution fractions from the rho1D4 immunoaffinity purification were concentrated to 0.75 mg/ml using a 10 kD MWCO filter column (Millipore, Billerica, MA) and then applied to the Ä kta system. After loading, the column was run with wash buffer at 1 ml/min and column flowthrough monitored via UV absorbance at 280 nm and 215 nm. Protein fractions were collected using an automated fraction collector. Peak fractions were then pooled, concentrated and subjected to SDS-PAGE and analysis via Sypro Ruby staining. Total protein concentration was measured using BCA assay (Pierce). Figure S1 PCR-based Gene Synthesis. Fabrication of the custom hOR17-4-rho gene was accomplished using a two-step PCR method using an overlapping oligonucleotide set designed using DNAWorks. (A) The first step is an assembly PCR in which all oligos function as both primer and template to effectively extend their 39 ends with successive PCR cycles. After many cycles of assembly, the full-length gene is constructed. However, the assembly reaction contains not only full-length product but also a mixture of all other extended oligos. Thus a second PCR reaction (B) is performed using a small amount of the assembly PCR reaction as template and the two terminal oligos (oligos A and L) as primers in order to selectively amplify the full-length gene product.